Llewelyn, Martin John; (2004) The influence of HLA class II polymorphisms on the properties of bacterial superantigens. Doctoral thesis (Ph.D), University College London.

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Abstract

The excessive and disordered immunological response triggered by bacterial superantigens has been implicated in the aetiology of a wide range of human diseases. The role of superantigens has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Since MHC class II presentation of superantigens to T cells is not MHC-restricted in the conventional antigen sense, the possibility that HLA polymorphisms could influence superantigenicity and thus, clinical susceptibility to the toxicity of individual superantigens, has received little attention. The work presented in this thesis focused principally on the streptococcal superantigen Streptococcal Pyrogenic Exotoxin A (SPEA) which utilises HLA-DQ but two staphylococcal superantigens, Staphylococcal Exotoxins A and B (SEA and SEB), were also studied. Using HLA class II homozygous B lymphoblastoid cell lines and HLA-DQ transfectants of Bare Lymphocyte Syndrome cell lines, HLA-DQ polymorphisms were shown to markedly influence SPEA binding assessed by flow cytometry. Specifically, SPEA binding by cell lines expressing HLA-DQA1*01 was up to ten fold higher than by cell lines expressing either DQA1*03 or *05. Using HLA-DQ molecules affinity purified from B lymphoblastoid cell lines, the impact of HLA-DQ polymorphisms on SPEA binding was confirmed in a cell free, ELISA based assay. Similar differences in SEA binding attributable to HLA-DR polymorphisms were observed in both the whole cell flow cytometry assay and the ELISA. Qualitative and quantitative differences were associated with differential HLA-DQ binding of SPEA and HLA-DR binding of SEA. Using splenocytes from the murine Vβ8.2 transgenic mouse strain DO11.10 as responder cells, both proliferation and cytokine release in response to SPEA presented by HLA-DQA1*01 were higher than when SPEA was presented by HLA-DQA1*03 or *05. Similar differences were observed using purified human T cells and using PBMCs from HLA-DQA1 homozygous healthy donors. Quantitiative differences in T cell response to SEA presented by different DR alleles were also observed. The Vβ specific changes in T cell repertoire that result from superantigen stimulation are regarded as being both the hall-mark of superantigenicity and defining of an individual superantigen. In the process of characterising the Vβ specific response to SPEA we noted that SPEA concentration has a profound effect on the repertoire of responding T cells. As the concentration of SPEA rises the T cell response broadens from Vβ14 to include Vβ12, 13.1 and 3. A similar effect was noted for SEA and SEB. Comparing the Vβ specific response in DQA1 homozygous healthy donors differences in Vβ repertoire were found comparing DQA1*01 donors with DQA1*03 donors. The work described in this thesis provides a molecular mechanism for observed differences in disease phenotype following infection by toxigenic strains of Staphylococci and Streptococci as well as the recent observation of an HLA association with predisposition to the severest manifestations of S. pyogenes infection. Given the widening perception of the importance of superantigens in disease pathogenesis, therapeutics and immunology the implications of these findings are very broad indeed. These data demonstrate that the HLA class II interaction with bacterial superantigens is an important host - pathogen interface where host polymorphisms may have driven the generation of diversity in superantigen producing strains of bacteria much as HLA class II diversity has arisen largely in response to infectious disease.

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